CN107488235B - A kind of preparation and application of new enhanced antigen joint polypeptid induction liver cancer-specific CTL cell - Google Patents

A kind of preparation and application of new enhanced antigen joint polypeptid induction liver cancer-specific CTL cell Download PDF

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CN107488235B
CN107488235B CN201710933388.4A CN201710933388A CN107488235B CN 107488235 B CN107488235 B CN 107488235B CN 201710933388 A CN201710933388 A CN 201710933388A CN 107488235 B CN107488235 B CN 107488235B
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rhil
liver cancer
cell
ctl
polypeptide
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CN107488235A (en
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闾军
陈辉
孙文峰
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Beijing Gene Qiming Biology Technology Co ltd
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Beijing Gene Qiming Biological Science And Technology Co Ltd
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Abstract

The present invention provides the preparations and application of a kind of new enhanced GPC3-AFP-NY-ESO-1 joint polypeptid induction liver cancer-specific CTL cell.The new liver cancer cells surface antigen polypeptide that present invention selection is filtered out from GPC3, AFP, NY-ESO-1 carries out sequential segments connection.GPC3, AFP, NY-ESO-1 polypeptide sequence newly filtered out is connected as CTL epitope peptide SEQ ID NO:4.The present invention establishes a kind of new method and means that efficient specific tumour killing is carried out for liver cancer cells, obtained liver cancer-specific killing CTL cell overcomes for tumor-killing inefficiency, key issues of incubation time is too long, it is capable of the effect of specificity enhancing liver cancer immunity treatment, there is cancer therapeutic applications prospect.

Description

A kind of system of new enhanced antigen joint polypeptid induction liver cancer-specific CTL cell Standby and application
Technical field:
The invention belongs to immunocyte preparation fields, and in particular to a kind of new enhanced GPC3-AFP-NY-ESO-1 connection Close the preparation method and application of polypeptid induction liver cancer-specific CTL cell.
Background technique:
China is global primary hepatoma (Primary Hepatocellular Carcinoma) high-incidence country, entirely Ball newly sends out about 1,000,000 people of liver cancer every year, and China accounts for 55%.China's cancer registry net statistics display in 2015, before 60 years old In males, the dead accounting highest of liver cancer.In recent years, although having there is some breakthroughs in the treatment, such as surgery hand Art, interventional therapy and transplanting, but the rapid progress of liver cancer, metastatic etc. are still the critical issue for needing to overcome.Tumour Immunization therapy has become treatment at present and eliminates the important means and method of tumour, will also become and treats the important of tumour from now on Direction.Intracorporal immune system mainly includes inherent immunity and adaptive immune system, and inherent immunity and adaptive immunity play The mode and means of effect are had nothing in common with each other, they are all played an indispensable role in the invasion for resisting tumour.It is controlled in tumour During treatment, present adoptive immunity cell therapy has been had been widely adopted, and adoptive immunity cell therapy mainly passes through external evoked The cell and cell factor for stimulating with cytotoxicity and killing ability with enorganic biological regulation system is activated generate, Enhance the differentiation capability of the specific cell of body killing tumour, killing tumor cell, simultaneously effective presses down on a cellular level The recurrence and transfer of tumour cell processed, and improve the autoimmunity function of tumor patient.
Clinically applying more now is the specific antibody for being directed to tumor targets, and the half-life period of antibody in vivo is shorter, Need to be spaced repeat administration, treatment cost is expensive and there are more side reactions.The method of immune cell therapy overcomes biography The shortcomings that treating is ruled, with developing deeply on cellular level and molecular level to the understanding of tumour immunity, loads tumour antigen Dendritic Cells (Dendritic Cells, DC) carry out sensitization autologous leukocytes, to obtain thin for tumour-specific The method of Cytotoxic T Lymphocytes (Cytotoxic T Lymphocyte, CTL), has become the important hand of immunization therapy Section and method.The specific CTL obtained by this method, can in vitro or internal massive amplification, and have immune Memorability, specific recognition tumor surface antigen, killing tumor cell.CTL needs specific tumor antigen and antigen presentation The costimulatory signal that cell (Antigen-presenting cell, APC) provides activates jointly, for tumour relative specific The screening of antigen becomes the emphasis of recent researches, how to filter out the tumor associated antigen that immunogenicity is strong, specificity is high (Tumor-associated antigen, TAA) tumour specific antigen (Tumor specific antigen, TSA) becomes Oncotherapy research worker needs the major issue solved.For the polypeptide immunotherapeutical vaccine of TAA or TSA, safe, It is easily prepared save, specificity it is high, can effectively killing tumor cell, become present widely used treatment method.
The activation of cytotoxic T lymphocyte needs APC to play the effect for presenting antigen, and APC can resist tumour correlation Original is degraded to polypeptide and is presented to T cell receptor (T Cell Receptor, TCR) progress after forming polypeptide-MHC compound Identification, so that activating cytotoxic T-lymphocyte, generates cell-cytotoxic reaction.Polypeptide vaccine is swollen with high dose and wide spectrum Tumor antigen polypeptide gives APC, combines itself and MHC molecule, can be presented to cytotoxic T cell to effective and wide spectrum.By In characteristics such as restricted, polypeptide vaccine less immunogenic of the tumor-antigen peptide by MHC molecule, and China HLA-A2 people Group's ratio is higher, and ratio is higher in liver cancer patient, therefore we are combined by force using new enhanced HLA-A2 class polypeptide antigen Antigen presentation function DC, can effectively improve immunogenicity, and inducing function is stronger, the more lethal cytotoxic T of type Lymphocyte.
Summary of the invention
The present invention provides a kind of new enhanced GPC3-AFP-NY-ESO-1 to combine polypeptid induction liver cancer-specific CTL The preparation method and application of cell.
The present invention provides a kind of new enhanced joint antigen polypeptide of induction liver cancer-specific CTL cell, according to The liver cancer-specific of GPC3, AFP, NY-ESO-1 are expressed and function difference, by amino acid sequence scoring and functional experiment, screening It is matched with HLA-A*0201 masculine liver cancer cell out and the liver of differential stimulus effect can be played for new undocumented three kinds Cancer cell surface antigens peptide sequence, and it is fabricated to joint polypeptide anti-liver cancer and anti-vaccine, this new joint polypeptide vaccine is compared to biography Hepatoma Vaccine of uniting has apparent advantage, the diversity that tumor cell surface antigen can be overcome to express, the difficult points such as inhomogeneity. The present invention also provides a kind of preparation method of enhanced liver cancer-specific CTL cell that induction is new and its in the treatment of liver cancer Using.
The new liver cancer cells surface antigen polypeptide that present invention selection is filtered out from GPC3, AFP, NY-ESO-1 carries out suitable The connection of sequence segment.By GPC3, AFP, NY-ESO-1 for newly filtering out, (its amino acid sequence is respectively: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) polypeptide sequence is connected as CTL epitope peptide SEQ ID NO:4.It is prepared using aforesaid way New polypeptide have induction be directed to hepatocellular carcinoma antigen epitope killing and immunological enhancement, it is this to liver cancer cells surface joint New polyvaccine ratio there is stronger killing function of tumor using traditional Antigenic Peptide stimulation.
The present invention is achieved by the following technical programs:
A kind of new enhanced liver cancer cell specificity combines polypeptide, amino acid sequence be the GPC3 newly filtered out, The combination of AFP, NY-ESO-1 polypeptide sequence.
GPC3 polypeptid acid sequence is AFP polypeptid acid sequence shown in SEQ ID NO:1 in the polypeptide composition It is shown in SEQ ID NO:2, NY-ESO-1 polypeptid acid sequence is shown in SEQ ID NO:3.
Further, the enhanced liver cancer cell specificity combines polypeptide, and amino acid sequence is SEQ ID NO:4 institute Show.
Further, the present invention provides enhanced liver cancer cell specificity joint polypeptides in preparation liver cancer cells Purposes in specific CTL inducer composition.
Invention also provides enhanced liver cancer cell specificity joint polypeptide preparations to have anti-liver cancer and anti-special The method of property CTL lymphocyte, the method comprises the following steps:
1) the peripheral blood mononuclear cells PBMC of HLA-A*0201 masculine liver cancer patient, adherent cell collecting, warp are separated RhGM-CSF and rhIL-4 Fiber differentiation, obtain non-mature dendritic cell；
2) continue the non-mature dendron shape obtained in Fiber differentiation step 1) using rhIL-2, rhIL-33 and rhTNF- α Cell obtains mature dendritic cell；
3) SEQ ID NO:4 polypeptide is added in the mature dendritic cell obtained into step 2), continues culture culture, Obtain the mature dendritic cell of sensitization；
4) mature dendritic cell for the sensitization that step 3) obtains is existed with the original non-adherent T lymphocyte separated Co-incubation in CTL special culture media is obtained with anti-liver cancer and anti-specific CTL lymphocyte.
Preferably, CTL culture medium used in step 4) contains the rhIL-2,2.5- that concentration is 100-500IU/ml The rhIL- of the rhIL-21 of the rhIL-15 of the rhIL-7 of 15ng/ml, 2.5-15ng/ml, 2.5-15ng/ml, 2.5-15ng/ml The anti-cd 3 antibodies of 23,0.5-5 μ g/ml；It is 300-500IU/ml's that the CTL culture medium more preferably used, which contains concentration, The rhIL-21 of the rhIL-15 of the rhIL-7 of rhIL-2,5-15ng/ml, 5-15ng/ml, 5-15ng/ml, 5-15ng/ml's The anti-cd 3 antibodies of rhIL-23,1-3 μ g/ml；The rhIL-2 for being further preferably 300-400IU/ml containing concentration, 10-15ng/ml's The rhIL-23 of the rhIL-21 of the rhIL-15 of rhIL-7,10-15ng/ml, 10-15ng/ml, 10-15ng/ml, 1-2 μ g/ml Anti-cd 3 antibodies；RhIL-2, the rhIL-7 of 10ng/ml for being most preferably 300IU/ml containing concentration, 10ng/ml's The rhIL-23 of the rhIL-21 of rhIL-15,10ng/ml, 10ng/ml, the anti-cd 3 antibodies of 1 μ g/ml.
Invention also provides it is above-mentioned prepare have anti-liver cancer and anti-specific CTL lymphocyte preparation liver cancer control Treat the purposes in drug.
In embodiments of the present invention, the CTL of joint antigen inducing peptide hepatocellular carcinoma antigen specificity, the joint are provided Antigenic Peptide is the antigen polypeptide association aggregation object in liver cancer cell specificity expression.The enhanced CTL of combination of the present invention is anti- Former peptide is GPC3, AFP, NY-ESO-1 polypeptide sequence connection polymer in liver cancer-specific expression.
In implementation method of the invention, the combined mode for combining Antigenic Peptide is specially the liver cancer cells antigen Tri- novel polypeptides of GPC3, AFP and NY-ESO-1 are linked in sequence, wherein the amino acid sequence of the GPC3 polypeptide is SEQ ID NO: Shown in 1, the amino acid sequence of the AFP polypeptide is shown in SEQ ID NO:2, and the amino acid sequence of the NY-ESO-1 polypeptide is Shown in SEQ ID NO:3.The mode of connection is sequential segments connection method.The amino acid sequence of joint polypeptide of the present invention is such as Shown in SEQ ID NO:4:
SEQ ID NO:4
N-VLLGLFSTI-FIYEIARRH-ITQCFLPVF-C
SEQ ID NO:1
N-VLLGLFSTI-C
SEQ ID NO:2
N-FIYEIARRH-C
SEQ ID NO:3
N-ITQCFLPVF-C
The liver cancer-specific CTL cell for the new joint polypeptid induction that the present invention is prepared, having largely enhances Anti-liver cancer and anti-cell fragmentation effect, it is many to secrete the more traditional antigen polypeptide enhancing of lethal cell factor IFN-γ ability, Its specificity enhancing for being directed to liver cancer cells inhibits the ability of hepatoma cell proliferation differentiation and kills ability better than tradition to it Antigen peptide vaccine.
Innovation and significance of the invention is embodied in: being established a kind of for the efficient specific tumour of liver cancer cells progress The new method and means of killing.The liver cancer-specific killing CTL cell that the present invention obtains overcomes for tumor-killing low efficiency Under, key issues of incubation time is too long, is capable of the effect of specificity enhancing liver cancer immunity treatment, before having cancer therapeutic applications Scape.
Detailed description of the invention
Fig. 1 new enhanced joint liver cancer-specific polypeptide sequence
Fig. 2 has the mature dendritic cell of sensitization and combines antigen inducing peptide liver cancer-specific CTL lymphocyte Preparation
Liver cancer-specific CTL effector cell's IFN-γ secretion ability of Fig. 3 epitope induction
Cytotoxicity in vitro ability of the specific CTL effector cell of Fig. 4 epitope induction to liver cancer cell lines HepG2
Cytotoxicity in vitro ability of the specific CTL effector cell of Fig. 5 epitope induction to liver cancer cell lines Huh7
Fig. 6: the specific CTL effector cell of epitope induction kills liver cancer cells experiment in animal body
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment.Experiment side in following embodiments Particular technique or condition person is not specified in method, then according to conventional laboratory conditions condition as described in " ATCC cell culture handbook ", and When having clearly stated Reagent Company's specification in embodiment, then to specifications proposed by condition carry out.Agents useful for same or Production firm person is not specified in instrument, and being can be with conventional products that are commercially available.
Reagent used in the embodiment of the present invention:
AIM V cell culture medium, ThermoFisher company
Lymphoprep separating liquid (instant), Axis-Shield company
CD3 monoclonal antibody, rhIL-2, rhIL-7, rhIL-15, rhIL-21, rhIL-23, rhIL-33, rhIL-4, RhGM-CSF, rhTNF- α, Peprotech company
Embodiment 1 synthesizes liver cancer-specific and combines polypeptide
HLA molecule determines more general with the polypeptide of specific length and affinity in the amino acid residue of specific position The affinity that peptide is identified by TCR.We pass through the database of SYFPEITHI and BIMAS, have carried out GPC3, AFP, NY-ESO-1 The prediction of polypeptide and MHC-I class molecule binding ability, and the screening that is compared and scores.We have obtained new having HLA Sequence SEQ ID NO:1, SEQ ID NO:2, the SEQ ID NO:3 of GPC3, AFP, NY-ESO-1 of high affinity molecule.It looks into Relative literature patent report is read, the above sequence is not published in correlative study.
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and the classical antigen that screening is obtained Amino acid sequence shown in peptide group (sequence has been reported) SEQ ID NO:5, commission Beijing SBS Genetech gene technology Co., Ltd use TETRASTMPeptide Synther is synthesized, and carries out purifying and purity analysis, mass spectrography using high performance liquid chromatography Carry out identification and molecular weight determination.The results show that synthesis polypeptide purity is higher than 95%, molecular weight is consistent with theoretical value.Institute is much Peptide is GMP grades.Specific synthesis step: 1. Solid phase peptide synthssis Fmoc method, basic solvent remove amido protecting group；2. Activator activates amino acid c-terminus, the monomer of activation and free amine group is carried out cross-linking reaction under crosslinking action, repeatedly Circulation is until peptide chain synthesis finishes.
2 peripheral blood mononuclear cells of embodiment (PBMC) is extracted
(1) early morning separates the anticoagulant venous blood of HLA-A*0201 masculine liver cancer patient 20ml.It is added in 50ml centrifuge tube 15ml Lymphoprep separating liquid.
(2) it is slowly added to isometric 0.9% sterile saline gentle inversion in anticoagulant venous blood three times, mixes well.
(3) blood after dilution is slowly added into lymphocyte separation medium surface layer using 2ml sterile dropper, after all moving into It is sure not to shake or overturns.
(4) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:0/0, room temperature centrifugation 30min。
(5) the extra blood plasma in top layer part is sucked out.Buffy coat is gently sucked out with 2ml aseptic straw, moves into new In 50ml centrifuge tube, the buffy coat in all pipes is sucked in the same 50ml centrifuge tube.0.9% sterile physiological salt is added Water is mixed well to 50ml.
(6) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:9/9 centrifugation, room temperature from Heart 10min.
(7) test tube is taken out, discards liquid completely.
(8) the AIM V culture medium that incubation is taken out from 37 DEG C of incubators, takes 2ml to mix well cell precipitation, pays attention to light Soft operation avoids generating bubble.Remaining culture medium is then added, mixes cell.
(9) cell suspension of mixing is moved into 75cm2Tissue Culture Flask shakes gently mixing, is placed in 37 DEG C of incubators Middle culture.
The preparation of 3 antigen presenting cell of embodiment
(1) PBMC for obtaining density-gradient centrifugation method is added in Tissue Culture Dish, 37 DEG C with the resuspension of AIM V culture medium, 5%CO2Culture.
(2) 3 as a child took out culture dish, shaked gently, and suspension cell is sucked out.
(3) the AIM V culture medium of the rhGM-CSF of rhIL-4,500IU/ml containing 500IU/ml are added into culture dish.
(4) respectively after incubation the 3rd day, the 5th day half amount change liquid, fresh rhIL-4,500IU/ containing 500IU/ml is added The rhGM-CSFAIM V culture medium of ml.
(5) it cultivates and rhTNF- α of final concentration of 10ng/ml, rhIL-2,10ng/ml of 300IU/ml is added within the 6th day The culture medium of rhIL-33.
(6) the 7th days harvest mature dendritic cells.
(7) mature dendritic cell is collected in centrifugation, and with the resuspension of AIM V culture medium, 10 μ g/ml amino acid sequence of final concentration is added It is classified as the joint antigen polypeptide of SEQ ID NO:4 or compares the culture of Antigenic Peptide group 6 hours.
(8) the free polypeptide of centrifugation removal, harvests the mature dendritic cell with sensitization.
Embodiment 4 combines the preparation of the enhanced liver cancer-specific CTL effector cell of polypeptid induction
(1) HLA-A*0201 Peripheral Blood of Patients with Hepatocellular Carcinoma is resuspended in through the isolated non-adherent suspension cell of 2 method of embodiment In AIM V culture medium, cell concentration is adjusted to 1 × 106A/ml is rich in T lymphocyte in this suspension.
(2) mature dendritic cell with sensitization for preparing embodiment 3 and T lymphocyte according to DC:T cell= After the ratio mixing of 1:10, rhIL-2, the rhIL-7 of 10ng/ml of final concentration 300IU/ml are added in AIM V culture medium, The CD3 antibody of the rhIL-23 of the rhIL-21 of the rhIL-15 of 10ng/ml, 10ng/ml, 10ng/ml, 1 μ g/ml continue culture 10 It, every 3 days half amounts change liquid, and cell is collected after 10 days up to the specific CTL effector cell of joint epitope induction.
Embodiment 5 combines liver cancer-specific CTL effector cell's IFN-γ secretion ability test experience of epitope induction
Use IFN- in real-time fluorescence quantitative PCR (real-time quantitative PCR, Q-PCR) detection cell γ secretion capacity.
The IFN-γ PCR primer sequence is as follows:
Forward:5 '-TCTGTGTGGATTGGTGGCTCTA-3 '
Reverse:5 '-CCTCGAACTTGGCGATGCT-3 '
Q-PCR reaction carries out on BIO-RAD IQ5 model quantitative PCR apparatus, reaction system are as follows:
cDNA 2μL
primer1(10μΜ)0.2μL
primer2(10μΜ)0.2μL
SYBR Green Realtime PCR Master 10μL
ddH2O 7.6μL
20 μ L of total system
Gene expression amount calculates the 2- Δ Δ CT method using optimization, and using GAPDH gene as internal reference, each sample is each 3 technologies of experimental setup repeat, and each processing is arranged 4 biology and repeats.
Experimental group are as follows: PBS control group；AFP antigen group；GPC3 antigen group；NY-ESO-1 antigen group；Joint Antigenic Peptide Group；Classical Antigenic Peptide group.
GPC3 antigen group: SEQ ID NO:1 sequence peptide fragment, 10 μ g/mL of final concentration；
AFP antigen group: SEQ ID NO:2 sequence peptide fragment, 10 μ g/mL of final concentration；
NY-ESO-1 antigen group: SEQ ID NO:3 sequence peptide fragment, 10 μ g/mL of final concentration；
Classical Antigenic Peptide group (sequence has been reported): SEQ ID NO:5 sequence peptide fragment, 10 μ g/mL of final concentration；
Joint Antigenic Peptide group: SEQ ID NO:4 sequence peptide fragment, 10 μ g/mL of final concentration.
Fig. 3's the result shows that, joint Antigenic Peptide group induction liver cancer-specific CTL lymphocyte IFN-γ secretion ability most By force, relatively traditional Antigenic Peptide and classics have reported that Antigenic Peptide group secretion inducing IFN-γ ability at least improves two to three times.
The liver cancer-specific CTL of the reinforced joint antigen polypeptide induction of embodiment 6 kills capacity experimental to liver cancer cells
Effector cell: the CTL that embodiment 4 obtains
Target cell 1: Hepatoblastoma cell line HepG2
Target cell 2: people's differentiated liver cancer system Huh7
It detects specific CTL and lactic dehydrogenase (Lactic is used to tumor cell killing potential experiment Dehydrogenase, LDH) method for releasing.The specific operation method is as follows:
(1) target cell concentration is adjusted to 1 × 105A/ml；
(2) target cell is transferred in 96 orifice plates according to 100 holes μ l/, three multiple holes of every group of setting.
(3) target cell Spontaneous release hole (negative control) is set.Effector cell is not added and only adds 100 μ l culture solutions.
(4) maximum relief hole (positive control) is set.Effector cell is not added and only adds 100 μ l, 10% NP40.
(5) 100 μ l of effector cell, effector cell: target cell=0:1 are added in each experimental port；1:1；3:1；10:1； 30:1；50:1.
(6) 37 degree of 5%CO2It cultivates to the corresponding time.
(7) culture solution supernatant is drawn, detection LDH numerical value calculates activity.Killing activity (%)=[(experimental group A value-is total certainly So release A value)/(the total Spontaneous release A value of maximum release group A value -)] × 100%.
Fig. 4 the result shows that, the specific CTL effector cell of epitope induction is to the Cytotoxicity in vitro energy of liver cancer cell lines HepG2 Power: target cell is people liver mother cell cancer HepG2 cell line, and effector cell is the specific CTL of epitope induction, thin according to effect Born of the same parents: target cell different proportion, that is, 0:1,1:1,3:1,10:1,30:1,50:1 carries out killing experiments.It was collected on cell in 12 hours It is clear to carry out lactic dehydrogenase (LDH) detection.Phosphate buffer (PBS) group, GPC3 polypeptide, AFP polypeptide, NY-ESO-1 are set Polypeptide, classical joint polypeptide group are as control.The results show that joint Antigenic Peptide group induction CTL can form the spy to target cell Opposite sex dissolution is better than each control group and classical Antigenic Peptide group to the fragmentation effect of HepG2, it was demonstrated that the induction of joint epitope CTL function ratio is stimulated more effective using traditional Antigenic Peptide.
Fig. 5 the result shows that, Cytotoxicity in vitro ability of the liver cancer-specific CTL effector cell to liver cancer cell lines Huh7: target is thin Born of the same parents are people's differentiated hepatocellular carcinoma Huh7 cell line, and effector cell is the specific CTL of liver cancer induction, according to effector cell: target Cell different proportion, that is, 0:1,1:1,3:1,10:1,30:1,50:1 carries out killing experiments.Collected in 12 hours cell conditioned medium into Row lactic dehydrogenase (LDH) detection.Be arranged phosphate buffer (PBS) group, GPC3 polypeptide, AFP polypeptide, NY-ESO-1 polypeptide, Classics joint polypeptide group is as control.The results show that joint epitope induction CTL can be formed to the specific molten of target cell Solution is better than each control group and classical Antigenic Peptide group to the fragmentation effect of Huh7, it was demonstrated that joint antigen inducing peptide CTL function ratio It is stimulated using traditional Antigenic Peptide more effective.
Killing tumor cells of hepatocellular carcinoma experiment in embodiment 7.CTL cell body
Select the hepatocellular carcinoma cells system HepG2-GFP of GFP fluorescent marker as tumour cell, subcutaneous implantation is in week old 8 weeks, immunodeficient mouse NOD-SCID back of mice of the weight between 20~30g temporally put tail after tumour grows up to Vein is adopted people's CTL cell, and fluorescence imaging observation tumour growth situation simultaneously measures tumor size.
The specific operation method is as follows:
(1) combine the enhanced liver cancer-specific CTL effector cell of polypeptid induction: the massive amplification that embodiment 4 obtains CTL cell, it is spare with physiological saline suspension before injection；
(2) after CTL cell infusion frequency is since 14 days 1 times a week, totally 4 weeks, 4 injections, negative control group was note 1% physiological saline group containing albumin is penetrated, positive controls are intraperitoneal injection adriamycin 2mg/kg, are injected into abdominal cavity after 14 days start Every 5 days 1 time；
(3) inject the determination of CTL cell dosage: on the basis of the adult of 60kg, CTL cell is in the expected dosage of clinic It is 1 × 109~1 × 1010A cell (about 1.6 × 107~1.6 × 108A cell/kg).It is with NOD-SCID mouse weight 30g Benchmark, clinical projected dose are equivalent to 5 × 105~5 × 106A cell/only.We are using high dose cell as test dose, often Mouse adopts 5 × 106A cell；
(4) volume (mean tumor volume) of tumour: during injection, caliper (calipers) is used 3 times a week The long axis and short axle of tumour are measured, and utilizes calculation formula below, measures the volume of tumour: V (mean tumor volume, mm3)=AB2/ 2 (A=long axis length, B=minor axis lengths).
Every group of 3 NOD-SCID mouse of experimental group.Specifically it is grouped as follows shown in table.
By Fig. 6 the results show that NOD-SCID mouse subcutaneous implantation HepG2-GFP is after tumour cell 14 days, tail vein is adopted The enhanced liver cancer-specific CTL effector cell of joint polypeptid induction, time point of adopting are respectively the 14th, 21,28,35 day, this Fluorescence imaging observes tumor size weekly afterwards, and measures gross tumor volume (Fig. 6 A).Such as Fig. 6 B, 6C the results show that CTL cell mistake After to after tumor-bearing mice, mouse tumor size is obviously reduced compared with Control negative control group, prompts the increasing of joint polypeptid induction Strong type liver cancer-specific CTL effector cell plays the role of apparent anti-liver cancer and anti-tumour cell in vivo.
The content for the publication listed in all this specification is included in this specification.In addition, those skilled in the art Member carries out the present invention it is appreciated that without departing substantially from technical scope described in the claims and substantive content A variety of different modifications and change are possible.The invention also includes these above-mentioned modifications and changes.
Sequence table
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Claims (8)

1. a kind of enhanced liver cancer cell specificity combines polypeptide, which is characterized in that its amino acid sequence such as SEQ ID NO:4 institute Show.
2. the enhanced liver cancer cell specificity joint polypeptide in claim 1 is preparing liver cancer cell specificity CTL inducer Purposes in composition.
3. using the enhanced liver cancer cell specificity joint polypeptide preparation in claim 1 there is anti-liver cancer and anti-specific CTL to drench The method of bar cell, which is characterized in that the method comprises the following steps:
1) the peripheral blood mononuclear cells PBMC of HLA-A*0201 masculine liver cancer patient, adherent cell collecting, through rhGM- are separated CSF and rhIL-4 Fiber differentiation, obtain non-mature dendritic cell；
2) using rhIL-2, rhIL-33 and rhTNF- α continue Fiber differentiation step 1) in obtain Dendritic Cells, obtain at Ripe Dendritic Cells；
3) the enhanced liver cancer cell specificity joint of claim 1 is added in the mature dendritic cell obtained into step 2) Polypeptide continues to cultivate, and obtains the mature dendritic cell of sensitization；
4) mature dendritic cell for the sensitization for obtaining step 3) is trained with the original non-adherent T lymphocyte separated in CTL Co-incubation in base is supported, is obtained with anti-liver cancer and anti-specific CTL lymphocyte.
4. according to the method described in claim 3, it is characterized in that, CTL culture medium used in step 4) contains concentration is The rhIL-15 of the rhIL-7 of the rhIL-2 of 100-500IU/ml, 2.5-15ng/ml, 2.5-15ng/ml, 2.5-15ng/ml's The anti-cd 3 antibodies of the rhIL-23 of rhIL-21,2.5-15ng/ml, 0.5-5 μ g/ml.
5. according to the method described in claim 4, it is characterized in that, CTL culture medium used in step 4) contains concentration is The rhIL-21 of the rhIL-15 of the rhIL-7 of the rhIL-2 of 300-500IU/ml, 5-15ng/ml, 5-15ng/ml, 5-15ng/ml, The anti-cd 3 antibodies of the rhIL-23 of 5-15ng/ml, 1-3 μ g/ml.
6. according to the method described in claim 5, it is characterized in that, CTL culture medium used in step 4) contains concentration is The rhIL- of the rhIL-15 of the rhIL-7 of the rhIL-2 of 300-400IU/ml, 10-15ng/ml, 10-15ng/ml, 10-15ng/ml The anti-cd 3 antibodies of 21,10-15ng/ml rhIL-23,1-2 μ g/ml.
7. according to the method described in claim 6, it is characterized in that, CTL culture medium used in step 4) contains concentration is The rhIL-21 of the rhIL-15 of the rhIL-7 of the rhIL-2 of 300IU/ml, 10ng/ml, 10ng/ml, 10ng/ml, 10ng/ml's RhIL-23, the anti-cd 3 antibodies of 1 μ g/ml.
8. according to what the method any in claim 3-7 prepared there is anti-liver cancer and anti-specific CTL lymphocyte to exist Prepare the purposes in cancer treatment drug.
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